Ionizing radiation (IR) can cause apoptosis in many cell types, mediated by generation of reactive oxidizing species (ROS) either directly by the IR or via indirect mechanisms. Much evidence indicates that IR-induced apoptosis results from damage to DNA, but there is also evidence suggesting that non-nuclear energy deposition (e.g., in plasma membrane) can initiate apoptosis. It is unclear whether non-nuclear initiated apoptosis involves DNA-dependent processes, what the nature of the specific apoptosis triggering species is, and what apoptotic pathway(s) are triggered by non-nuclear IR. Hence, the overall goal of this project is to increase understanding of mechanisms of apoptosis initiation by IR in non-nuclear and nuclear sites. A combination of two unique approaches for generating ROS in selected subcellular regions will be used. Ionizing radiation microbeams will be used to irradiate selectively nuclear and non-nuclear cell regions. This will be complemented by use of photoactive agents that selectively produce OH, OR and OOR as a means to "mimic" IR. The project has three specific aims: (1) Measure the induction of apoptosi: after non-nuclear and nuclear IR using microbeams and compare that efficacy with apoptosis induced by photoactive agents that produce OH, OR and OOR. We will test the hypothesis that ROS produced by IR in non-nuclear regions can trigger apoptosis, but with lower efficiency than IR/ROS in the nucleus. (2) Determine the relative contributions of different apoptotic pathways triggered by spatially restricted IR/ROS in nuclear versus non-nuclear cell regions to overall apoptosis from IR. (3) Test the hypothesis that nonnuclear IR/ROS in target cells can cause apoptosis in neighboring, untreated (bystander) cells. The mechanistic insight gained in these studies should increase understanding of complex, but clinically important, questions about the contribution of apoptosis to tumor cure from radiation therapy or to cancer development, and help suggest means to manipulate apoptotic pathways for therapeutic gain.